Biosynthesis of extracellular matrix molecules was studied using cultured monkey retinal pigment epithelial (RPE) cells. In one set of experiments, native interphotoreceptor matrix (IPM) was labeled in vivo to parallel the apical expression of putative IPM components by cultured RPE. By polyacrylamide gel electrophoresis and subsequent autoradiography, we found that cultured RPE produces several proteins whose electrophoretic mobility coincides with those of proteins in vivo. Therefore, cultured RPE may be a source of previously unidentified IPM constituents. Also shown was cultured RPE synthesis of two well-characterized basement membrane components, the attachment glycoprotein laminin and heparan sulfate proteoglycan (HSPG). While the depositon of the former was polarized to the basal side of the cells, the latter was released in insoluble form as part of the extracellular matrix substrate and secreted in soluble form into the culture media. Cultured RPE cells do not proteolytically "chip" the precursor to the core protein of HSPC, but instead release an intact proteoglycan with glycosaminoglycan chains attached to the full-size 400 kD protein moiety.